Although thirty years of gerontologic investigation utilizing various cell culture model systems has provided many tantalizing clues regarding the general loss of cellular response to physiologic signals, the mechanism of cellular aging remains quite obscure. This competitive renewal application proposes to extend studies undertaken two years ago, employing a powerful combination of molecular biology and cell culture, to advance our understanding of age-associated changes in cellular proliferative capacity and differentiated function. The proposal draws upon the different perspectives and expertise of four principal investigators and on extensive data generated during the first funding period. Dr. Gilchrest's project will use early passage human keratinocytes and fibroblasts derived from skin biopsies of healthy newborn, young adult and old adult donors to determine whether age-associated loss of growth factor responsiveness is attributable to reduced expression of genes whose products are required for cell division and/or to a more advanced state of cellular differentiation. Dr. Campisi's project will use human fetal lung fibroblasts to define the mechanisms by which changes in the expression of pHE-7, histone, c-fos, and ornithine decarboxylase occur in late passage WI-38 cells. Dr. Miller's project will seek to define further the biochemical defects that underlie changes in calcium-mediated signal transduction and gene expression that occur following mitogenic stimulation of old vs. young murine lymphocytes. Dr. Dobson, a new member of the program project team, proposes to study differentiation-associated gene expression in the 3T3F442A murine preadipocyte cell line aged in vitro and in short-term adipocyte cultures obtained from young, old, and old diet-restricted mice. These separate projects will share molecular biology and administrative core facilities and will interact extensively with regard to both technical and conceptual issues. It is anticipated that the proposed studies will provide major new insights into cellular senescence generally, as well as age-associated changes in the skin, immune system, and adipose tissue of intact organisms. Furthermore, the proposed studies will permit corroboration of findings among widely differing model systems.